This invention relates to biologically active factors. In particular, the invention relates to inhibitors of hematopoietic progenitor cell proliferation and differentiation.
There are several known stimulators and inhibitors of hematopoietic progenitor cell proliferation and differentiation. For instance, regulatory molecules termed colony-stimulating factors (CSF) stimulate clonal macrophage and/or granulocyte expansion and differentiation from committed hematopoietic progenitor cells. Broxmeyer, Int. J. Cell Cloning, 4, 378-405 (1986); Broxmeyer and Williams, CRC Crit. Rev. Oncol./Hematol., 8, 173-226 (1988). Other substances are known which inhibit hematopoietic progenitor cell proliferation and differentiation.
One such inhibitor is lactoferrin (LF). In vitro. LF is reported to inhibit the production by mononuclear cells of CSF's which stimulate granulocyte and macrophage colony formation. Broxmeyer et al., J. Exp. Med., 148, 1052-1067 (1978); Pelus et al., J. Exp. Med., 150, 277-92 (1979); Broxmeyer et al., Blood, 55, 324-33 (1980); Bagby et al., J. Clin. Invest., 68, 56-63 (1981); Pelus et al., Cell Tissue Kinet., 14, 515-26 (1981); Broxmeyer and Platzer, J. Immunol., 133, 306-314 (1984); Fletcher and Willars, Blood Cells, 11, 447-54 (1986). LF may act directly on the monocytes to inhibit production of CSF's, but evidence has also been presented that LF inhibits the production of CSF by suppressing the production by monocytes of a monokine (e.g., interleukin-1) that recruits other cells (such as skin-derived human fibroblasts) to produce CSF. Bagby et al., J. Clin. Invest., 71, 340-44 (1983); Zucali et al., Blood, 74, 1531-36 (1989). See also. Broxmeyer et al., Blood Cells, 13, 31-48 (1987).
In vivo, iron-saturated human LF decreases the cycling status and the absolute numbers of murine marrow and spleen granulocyte-macrophage (CFU-GM), erythroid (BFU-E) and multipotential (CFU-GEMM) progenitor cells, whereas heat-inactivated LF does not. Gentile and Broxmeyer, Blood, 61, 982-93 (1983); Broxmeyer et al., Blood Cells, 13, 31-48 (1987). It is believed that the myelosuppressive effects of LF in vivo may result indirectly from the decreased release of growth factors. Broxmeyer et al., Blood Cells, 13, 31-48 (1987); Broxmeyer et al., J. Clin. Invest., 79, 721-30 (1987).
Acidic isoferritin (AIF) has been reported to inhibit colony formation by CFU-GM, BFU-E and CFU-GEMM progenitor cells. Broxmeyer et al., J. Exp. Med., 153, 1426-44 (1981); Broxmeyer et al., Blood, 60, 595 (1982); Broxmeyer et al., Blood Cells, 10, 397-426 (1984). In mice, human AIF suppresses the numbers of CFU-GM, BFU-E and CFU-GEMM per femur, decreases the cycling status of these cells and decreases the number of nucleated cells in the bone marrow and peripheral blood. Broxmeyer et al., Blood Cells, 10, 397-426 (1984); Broxmeyer et al., Blood, 73, 74-79 (1989). There is evidence that the inhibitory activity resides in a subpopulation of AIF molecules. Broxmeyer et al., Blood Cells, 10, 397-426 (1984). Some cell lines producing AIF are also reported to produce other unidentified inhibitory activities. Broxmeyer et al., Blood, 60, 595-607 (1982).
In vitro, prostaglandin E (PGE) inhibits the proliferation of CFU-GM, selectively inhibits CFU-GM in S-phase of the cell cycle and demonstrates a preferential inhibitory effect on monocytopoiesis. Pelus et al., J. Exp. Med., 150, 277-92 (1979); Pelus et al., Cell Tissue Kinet., 14, 515-26 (1981); Pelus and Gentile, Blood, 71, 1633-40 (1988). Intravenous administration of prostaglandin E.sub.2 (PGE.sub.2) to rebounding or normal mice results in significant inhibition of total nucleated cellularity and absolute number of CFU-GM in spleen and bone marrow, preferential inhibition of monocyte production, and selective inhibition of CFU-GM in S-phase of the cell cycle. Gentile et al., Blood, 62, 1100-1107 (1983); Gentile and Pelus, Exp. Hematol., 15, 119-256 (1987); Pelus and Gentile, Blood, 71, 1633-40 (1988).
Pelus and Gentile, Blood, 71, 1633-40 (1988) reports that the intravenous injection of PGE.sub.2 into intact mice induces a suppressor mechanism capable of suppressing CFU-GM Inhibition of CFU-GM proliferation was observed using either bone marrow or spleen cells from such mice or by using conditioned medium (CM) prepared by culturing the bone marrow or spleen cells. The article further teaches that the CM suppresses BFU-E, as well as CFU-GM, indicating that the inhibitory activity is not restricted to granulopoiesis. The molecular weight of the inhibitory activity was estimated to be about 4-6,000.
Gentile and Pelus, The Journal of Immunology, 141, 2714-20 (1988) further describes this inhibitory activity found in CM prepared by culturing cells from mice that have been injected with PGE.sub.2. The article teaches that morphological analysis of CFU-GM revealed an equivalent inhibition of monocyte, monocyteneutrophil and neutrophil CFU-GM in contrast to the preferential effect of PGE.sub.2 on monocytopoiesis. The inhibitory activity found in the CM was lost when the CM was treated with heat (56.degree. C. for 30 minutes or 100.degree. C. for 5 minutes), trypsin, chymotrypsin, pronase, and neuraminidase, but not when treated with lipase. The article reports that acrylamide-agarose gel filtration of the bone marrow CM revealed an active inhibitory fraction in the range of 5.5 to 8.0 kDa.
Taniguchi et al., Blood, 73, 907-913 (1989) reports that CM prepared by culturing cells taken from patients suffering from hairy cell leukemia inhibits the growth of granulocyte and erythrocyte colony forming cells. However, the hairy cell CM did not inhibit colony formation by all CSF's that stimulate granulopoiesis, and the erythrocyte precursors were also heterogeneous in their response to it. The article reports that the inhibitory activity in the CM is nondialyzable, fairly stable to heat treatment and destroyed by treatment with trypsin. The CM was fractionated, and activity against granulopoiesis was found in fractions having molecular weights in the range of 4,000-8,000 and over 160,000. Using another fractionation method, the inhibitory activity against granulopoiesis was found in only one peak of 5,000-6,000 daltons. The characteristics of the inhibitory activity in the hairy cell CM were compared to those of other inhibitors of hematopoiesis in the Discussion section of the Taniguchi et al. article.
U.S. Pat. No. 4,384,991 describes an inhibitor of the proliferation of normal and leukemic myeloid cells. The inhibitor is purified from an extract of white blood cells (granulocytes) isolated from blood or from an extract of an animal organ containing granulocytes. The patent reports that the inhibitor is a polypeptide and that its amino acid composition is Tau.sub.1, Asx.sub.1, Ser.sub.2, Thr.sub.1, Glx.sub.3, Gly.sub.2, Ala.sub.1 (PO.sub.4).sup.2-. From this amino acid composition, the molecular weight can be calculated to be about 1360. The patent further reports that the inhibitor's point of attack is the G.sub.1 phase of the cell cycle and that the inhibitor is thermostable.
Yet another inhibitor is described in Frindel and Guigon, Exp. Hemat., 5, 74-76 (1977). The inhibitor is a cell-free extract of fetal calf bone marrow. The article reports that the extract inhibits the proliferation of bone marrow colony forming cells in irradiated mice and that the inhibitor may have a rather low molecular weight since it is dialyzable.
Wright and Lorimore, Cell Tissue Kinet., 20, 191-203 (1987) teaches that medium conditioned by normal murine bone marrow cells contains an inhibitor of hematopoietic spleen colony-forming cell proliferation. The inhibitory activity can be concentrated in a nominal 50-100,000 molecular weight fraction.
Finally, Rogers et al., Cellular Immunology, 50, 82-93 (1980) describes a factor capable of nonspecifically suppressing phytohemagglutinin lipopolysaccharide and phytohemagglutinin lipopolysaccharide-induced mitogenesis. The factor was prepared by culturing glass-adherent T cells in the presence of PGE.sub.2. The article teaches that the culture supernatants contain at least two suppressors with approximate molecular weights of 35,000 and 5,000. The inhibitory activity was resistant to boiling and treatment with RNase and DNase, but was sensitive to treatment with proteases.